The invention relates to the identification of expression profiles and the nucleic acids involved in colorectal cancer, and to the use of such expression profiles and nucleic acids in diagnosis and prognosis of colorectal cancer. The invention further relates to methods for identifying and using candidate agents and/or targets which modulate colorectal cancer.
Colorectal cancer is a significant cancer in Western populations. It develops as the result of a pathologic transformation of normal colon epithelium to an invasive cancer. There have been a number of recently characterized genetic alterations that have been implicated in colorectal cancer, including mutations in two classes of genes, tumor-suppressor genes and proto-oncogenes, with recent work suggesting that mutations in DNA repair genes may also be involved in tumorigenesis. For example, inactivating mutations of both alleles of the adenomatous polyposis coli (APC) gene, a tumor suppressor gene, appears to be one of the earliest events in colorectal cancer, and may even be the initiating event. Other genes implicated in colorectal cancer include the MCC gene, the p53 gene, the DCC (deleted in colorectal carcinoma) gene and other chromosome 18 q genes, and genes in the TGF-xcex2 signalling pathway. For a review, see Molecular Biology of Colorectal Cancer, pp238-299, in Curr. Probl. Cancer, September/October 1997.
Imaging of colorectal cancer for diagnosis has been problematic and limited. In addition, dissemination of tumor cells (metastases) to locoregional lymph nodes is an important prognostic factor; five year survival rates drop from 80 percent in patients with no lymph node metastases to 45 to 50 percent in those patients who do have lymph node metastases. A recent report showed that micrometastases can be detected from lymph nodes using reverse transcriptase-PCR methods based on the presence of mRNA for carcinoembryonic antigen, which has previously been shown to be present in the vast majority of colorectal cancers but not in normal tissues. Liefers et al., New England J. of Med. 339(4):223 (1998).
Thus, methods that can be used for diagnosis and prognosis of colorectal cancer would be desirable. While academia and industry has made an effort to identify novel sequences, there has not been an equal effort exerted to identify the function of the novel sequences in disease states of concern, such as cancer. For example, databases show the sequence for accession numbers AA331393, N95719, Al1346620, AA411502, and AF179224, and the later has been identified as a transmembrane serine protease 3, but there is limited data correlating these sequences with a disease state. Further provided are methods that can be used to screen candidate bioactive agents for the ability to modulate colorectal cancer. Additionally, provided herein are molecular targets for therapeutic intervention in colorectal and other cancers.
The present invention provides methods for screening for compositions which modulate colorectal cancer. In one aspect, a method of screening drug candidates comprises providing a cell that expresses an expression profile gene or fragments thereof. Preferred embodiments of the expression profile genes as described herein include the sequence comprising CGA7, or a fragment thereof. Other preferred embodiments include the sequence comprising CJA8, or a fragment thereof. The method further includes adding a drug candidate to the cell and determining the effect of the drug candidate on the expression of the expression profile gene.
In one embodiment, the method of screening drug candidates includes comparing the level of expression in the absence of the drug candidate to the level of expression in the presence of the drug candidate, wherein the concentration of the drug candidate can vary when present, and wherein the comparison can occur after addition or removal of the drug candidate. In a preferred embodiment, the cell expresses at least two expression profile genes. The profile genes may show an increase or decrease.
Also provided herein is a method of screening for a bioactive agent capable of binding to a colorectal cancer modulating protein (CCMP) or a fragment thereof, the method comprising combining the CCMP or fragment thereof and a candidate bioactive agent, and determining the binding of the candidate agent to the CCMP or fragment thereof. In a preferred embodiment, the CCMP is CGA7. In another preferred embodiment, the CCMP is CJA8.
Further provided herein is a method for screening for a bioactive agent capable of modulating the bioactivity of a CCMP or a fragment thereof. In one embodiment, the method comprises combining the CCMP or fragment thereof and a candidate bioactive agent, and determining the effect of the candidate agent on the bioactivity of the CCMP or the fragment thereof. In a preferred embodiment, the CCMP is CGA7. In another preferred embodiment, the CCMP is CJA8.
Also provided herein is a method of evaluating the effect of a candidate colorectal cancer drug comprising administering the drug to a transgenic animal expressing or over-expressing a CCMP or a fragment thereof, or an animal lacking a CCMP for example as a result of a gene knockout. In a preferred embodiment, the CCMP is CGA7. In another preferred embodiment, the CCMP is CJA8.
Additionally, provided herein is a method of evaluating the effect of a candidate colorectal cancer drug comprising administering the drug to a patient and removing a cell sample from the patient. The expression profile of the cell is then determined. This method may further comprise comparing the expression profile to an expression profile of a healthy individual.
Furthermore, a method of diagnosing colorectal cancer is provided. The method comprises determining the expression of a gene which encodes CGA7 or a fragment thereof, in a first tissue type of a first individual, and comparing this to the expression of the gene from a second unaffected individual. A difference in the expression indicates that the first individual has colorectal cancer.
In another embodiment, the method comprises determining the expression of a gene which encodes CJA8 or a fragment thereof, in a first tissue type of a first individual and comparing this to the expression of the gene from a second unaffected individual. A difference in the expression indicates that the first individual has colorectal cancer.
In another aspect, the present invention provides an antibody which specifically binds to CGA7, or a fragment thereof. Preferably the antibody is a monoclonal antibody. The antibody can be a fragment of an antibody such as a single stranded antibody as further described herein, or can be conjugated to another molecule. In one embodiment, the antibody is a humanized antibody.
In another aspect, the present invention provides an antibody which specifically binds to CJA8, or a fragment thereof. Preferably the antibody is a monoclonal antibody. The antibody can be a fragment of an antibody such as a single stranded antibody as further described herein, or can be conjugated to another molecule. In one embodiment, the antibody is a humanized antibody.
In one embodiment a method for screening for a bioactive agent capable of interfering with the binding of CGA7 or a fragment thereof and an antibody which binds to said CGA7 or fragment thereof is provided. In a preferred embodiment, the method comprises combining CGA7 or a fragment thereof, a candidate bioactive agent and an antibody which binds to said CGA7 or fragment thereof. The method further includes determining the binding of said CGA7 or fragment thereof and said antibody. Wherein there is a change in binding, an agent is identified as an interfering agent. The interfering agent can be an agonist or an antagonist. Preferably, the antibody as well as the agent inhibits colorectal cancer.
In another embodiment a method for screening for a bioactive agent capable of interfering with the binding of CJA8 or a fragment thereof and an antibody which binds to said CJA8 or fragment thereof is provided. In a preferred embodiment, the method comprises combining CJA8 or a fragment thereof, a candidate bioactive agent and an antibody which binds to said CJA8 or fragment thereof. The method further includes determining the binding of said CJA8 or fragment thereof and said antibody. Wherein there is a change in binding, an agent is identified as an interfering agent. The interfering agent can be an agonist or an antagonist. Preferably, the antibody as well as the agent inhibits colorectal cancer.
In one aspect of the invention, a method for inhibiting the activity of a colorectal cancer modulating protein are provided. The method comprises binding an inhibitor to the protein. In a preferred embodiment, the protein is CGA7. In another preferred embodiment, the protein is CJA8.
In another aspect, the invention provides a method for neutralizing the effect of a colorectal cancer modulating protein. The method comprises contacting an agent specific for the protein with the protein in an amount sufficient to effect neutralization. In a preferred embodiment, the protein is CGA7. In another preferred embodiment, the protein is CJA8.
In a further aspect, a method for treating or inhibiting colorectal cancer is provided. In one embodiment, the method comprises administering to a cell a composition comprising an antibody to CGA7 or a fragment thereof. In another embodiment, the method comprises administering to a cell a composition comprising an antibody to CJA8 or a fragment thereof. In one embodiment, the antibody is conjugated to a therapeutic moiety. Such therapeutic moieties include a cytotoxic agent and a radioisotope. The method can be performed in vitro or in vivo, preferably in vivo to an individual. In a preferred embodiment the method of inhibiting colorectal cancer is provided to an individual with such cancer.
As described herein, methods of treating or inhibiting colorectal cancer can be performed by administering an inhibitor of CGA7 activity to a cell or individual. In one embodiment, a CGA7 inhibitor is an antisense molecule to a nucleic acid encoding CGA7. Alternatively, methods of treating or inhibiting colorectal cancer can be performed by administering an inhibitor of CJA8 activity to a cell or individual. In one embodiment, a CJA8 inhibitor is an antisense molecule to a nucleic acid encoding CJA8.
Moreover, provided herein is a biochip comprising a nucleic acid segment which encodes CGA7, or a fragment thereof, wherein the biochip comprises fewer than 1000 nucleic acid probes. Also provided herein is a biochip comprising a nucleic acid segment which encodes CJA8, or a fragment thereof, wherein the biochip comprises fewer than 1000 nucleic acid probes. Preferably a biochip includes at least two nucleic acid segments.
Also provided herein are methods of eliciting an immune response in an individual. In one embodiment a method provided herein comprises administering to an individual a composition comprising CGA7 or a fragment thereof. In another aspect, said composition comprises a nucleic acid comprising a sequence encoding CGA7 or a fragment thereof.
In another embodiment a method provided herein comprises administering to an individual a composition comprising CJA8 or a fragment thereof. In another aspect, said composition comprises a nucleic acid comprising a sequence encoding CJA8 or a fragment thereof.
Further provided herein are compositions capable of eliciting an immune response in an individual. In one embodiment, a composition provided herein comprises CGA7 or a fragment thereof and a pharmaceutically acceptable carrier. In another embodiment, said composition comprises a nucleic acid comprising a sequence encoding CGA7 or a fragment thereof and a pharmaceutically acceptable carrier.
In one embodiment, a composition provided herein comprises CJA8 or a fragment thereof and a pharmaceutically acceptable carrier. In another embodiment, said composition comprises a nucleic acid comprising a sequence encoding CJA8 or a fragment thereof and a pharmaceutically acceptable carrier.
Other aspects of the invention will become apparent to the skilled artisan by the following description of the invention.